Several essential properties of tyrosine hydroxylase were investigated in this laboratory. Tyrosine hydroxylase was purified to apparent homogeneity from rat pheochromocytoma cells, and many of the biochemical and physical properties of this tumor enzyme were characterized. In addition, rat pheochromocytoma tyrosine hydroxylase was cloned, expressed in E. coli, and subsequently purified to homogeneity. The pure recombinant enzyme exhibited many of the kinetic properties of native, activated tyrosine hydroxylase. The nature of this activation is currently under investigation. Other studies explored the mechanisms of tyrosine hydroxylase phosphorylation and dephosphorylation in intact rat striatal synaptosomes. Earlier evidence unveiled a pathway of dephosphorylation for tyrosine hydroxylase which was markedly stimulated by tetrahydrobiopterin in situ. This effect of tetrahydrobiopterin was shown to be specific and concentration dependent. Furthermore, the effect was observed following incubation of synaptosomes with cAMP, but not calcium ionophores or high potassium, and the response to tetrahydrobiopterin was eliminated by okadaic acid. These findings suggest that the action of tetrahydrobiopterin is mediated by a protein phosphatase of type 2A and is directed at sites that are phosphorylated by c-AMP-dependent protein kinase. Current studies are attempting to identify the specific phosphoamino acid target sites for tetrahydrobioptern.